The goal of this proposal is to develop methods to target light-activated proteins, such as Channel Rhodopsin II (ChR2) and Halorhodopsin (NpHR), to specific subcellular compartments in neurons. Both ChR2 and NpHR generate electrical currents in response to light: ChR2, an ion channel, passes depolarizing cation current in response to blue light, whereas NpHR, a pump, generates hyperpolarizing Cl- currents in response to yellow light. Neurons expressing these proteins can be efficiently excited or inhibited with light. ChR2 and NpHR can thus be expressed in specific neuronal populations to determine if activity in these cells is sufficient and necessary to drive a function, for example a behavior. ChR2 has been used to map synaptic circuits in brain slices, by combining patch clamp recording of postsynaptic cells with stimulation of presynaptic neurons expressing ChR2. ChR2 can also be used to stimulate neurons in a spatial pattern in vivo, for example to determine motor maps. A major limitation to the use of light-activated proteins in circuit mapping is their tendency to localize nonspecifically to different neuronal compartments. ChR2 and NpHR appear to be expressed equally well in axons and dendrites. In most neural tissues, dendrites and local and long-range axons are intermingled. The presence of these proteins in axons means that photostimulation can have non-local effects. Thus, it is virtually impossible to stimulate dendrites from one ChR2-positive cell without also stimulating neighboring ChR2-positive axons that can arise from distant and functionally unrelated neurons. Similarly, action potential propagation can be blocked by photostimulation of NpHR-positive axons. It is therefore of great interest to generate versions of NpHR and ChR2 that can be excluded from axons, by targeting them to dendrites and somata. In other applications it is advantageous to specifically target light- activated proteins to axons. Here we propose to generate peptides encoding signals that target light-activated proteins to specific subcellular compartments allowing neurons to be activated or inhibited for neural circuit analysis. PUBLIC HEALTH RELEVANCE: Light activated proteins have the potential to be used for treatment of neurological diseases such as depression and epilepsy. The technology developed in this grant will make the actions of light activated proteins both more precise and less likely to produce unwanted effects.